Isolation,Identification and Genomic Characterization of A Multi-lineage Recombinant NADC34-Iike PRRSV
In this study,lung tissue and serum samples of piglets suspected to be infected with PRRSV associated abortion were collected from large-scale pig farms in Gansu Province,China in September 2021.After ORF5 gene characteristic RT-PCR detection and sequencing,the virus was isolated with PAM cells.The strain was identified and isolated by indirect immunofluorescence assay and pathological electron microscopy sections of infected cells.The whole genome sequence of PRRSV isolates was amplified by RT-PCR.The genome characteristics and recombination were compared and analyzed with biological software.The results showed that the nucleotide and amino acid homology between the ORF5 gene and that of NADC34 strain was 94.9％and 95.5％,respectively.Indirect immunofluorescence showed that the virus could react with PRRSV N protein monoclonal antibody(SR30).Virions with diameter of 50-80 nm and total genome size of 15078 bp were observed in infected cells under electron microscopy.The isolate was named PRRSV GSJT/9/2021 strain.The nucleotide homology between the isolates and representative strains VR-2332,JXA1,HuN4,NADC30 and NADC34 were 85.2％,86.4％,86.5％,88.7％and 85.9％,respectively.Phylogenetic trees were constructed based on the whole genome of GSJT/9/2021 and ORF5 gene respectively to type the isolates.The results showed that the whole genome of GSJT/9/2021 was closely related to NADC30-like strains and belonged to the same cladistic branch.The phylogenetic tree of ORF5 gene showed that GSJT/9/2021 was closely related to NADC34-like strain.ORF5 and Nsp2 showed the highest degree of variation in the genome of the isolates.Four glycosylation sites in GP5 protein showed an additional glycosylation site 57th compared with NADC34 strain prevalent in China,and unique mutations(H38Q,L39F)appeared at the 38th and 39th spots of decoy epitopes 37-45 aa.131 discontinuous amino acids(323-433,483,504-522)were absent in Nsp2 protein.Recombination analysis showed that GSJT/9/2021 strain was derived from a complex recombination mutation between NADC30-like PRRSV,HUN4 and IA/2014/NADC34,and the recombination sites were located at the amino terminus of NSP4,NSP9,GP2a and M,respectively.The positive rate of PRRSV N protein antibody in 107 sera was 98.2％.In summary,PRRSV GSJT/9/2021 is a recombinant strain with multi-lineage recombination and mutation,and in-depth analysis of genetic variation and growth characteristics of the recombinant strain is of important guiding significance for prevention and control of PRRS in Gansu Province,China.
Porcine reproductive and respiratory syndrome virus(PRRSV)Multilineage recombinant strainIsolation and identificationBiological characteristicsCell tropism
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